Green fluorescent protein as a tool to study Glut4 trafficking

Campbell, Lachlan Clive (1999) Green fluorescent protein as a tool to study Glut4 trafficking. PhD thesis, University of Glasgow.

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Abstract

Glut4 is the principal glucose transporter expressed in the peripheral tissues. In response to insulin, Glut4 moves from an intracellular compartment to the plasma membrane in a process called translocation. Once at the cell surface, Glut4 transports glucose across the plasma membrane into the cell and thus regulates whole body glucose homeostasis. At the molecular level we tagged Glut4 with Green Fluorescent Protein (GFP) in order to glean further insight into the translocation process. The native fluorescence of GFP then allowed us to analyse the trafficking of GFP-tagged Glut4 chimeras in real time using confocal microscopy. Glut4-GFP expressed in 3T3-L1 fibroblasts or fully differentiated adipocytes, displayed an intracellular distribution that only partially overlapped with the early endosomal compartment. However, Glut4-GFP constructs that carried mutations in N- and C- terminus targeting motifs were localised to the plasma membrane. Time lapse confocal analysis revealed the dynamic nature of Glut4-GFP-containing vesicles in the basal state. When Glut4~GFP-expressing adipocytes were treated with insulin, Glut4-GFP translocated to the plasma membrane. Translocation was rapid, analogous to endogenous Glut4 translocation in 3T3-L1 adipocytes. However, upon insulin removal, Glut4-GFP failed to reinternalise. We suggest that this failure to reinternalise, while recycling normally in the basal state, reflects a Glut4-specific reinternalisation mechanism following insulin-stimulated translocation. Glut4-GFP exhibited a similar intracellular distribution when expressed in L6 muscle cells. Nitric oxide (NO) donors stimulate glucose uptake into L6 muscle cells. Using inhibitors of the nitric-oxide signalling pathway and Glut4-GFP we investigated the mechanism by which NO-donors increase glucose transport into L6 cells. Recent research has also implicated ADP- Ribosylation Factors (ARFs) in insulin-stimulated Glut4 translocation. ARFs are small molecular weight GTPases. By site-directed mutagenesis we endeavoured to change the specificity of the ARFS and ARF6 isoforms from GTP to XTP. These mutations would allow us to control ARF5/6 function in stable 3T3-L1 cell lines. Our goal is then to analyse the contribution of ARFS and ARF6 to insulin stimulated Glut4-GFP translocation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Gwyn Gould
Keywords: Molecular biology
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-71820
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 17 May 2019 09:31
URI: http://theses.gla.ac.uk/id/eprint/71820

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